This application seeks support for molecular and genetic analyses of the first group II intron, L1.1trB, shown to be functional in vivo in prokaryotes. Group II introns are often found in organelles of eukaryotes and splice by a mechanism similar to that of spliceosomal introns in eukaryotic nuclear genes. They are also mobile elements which may translocate in genomes by novel retrotransposition mechanisms. Thus the molecular biology of these elements is relevant to the understanding of gene regulation and evolution in eucaryotes and viruses. L1.1trB was discovered in the laboratory of the PI in the course of collaborative research with the coinvestigator on conjugative plasmid transfer of the Lactococcus lactis plasmid pRSO1. Splicing of this intron is required for expression of a functional gene product from the exon gene, ltrB, encoding a conjugative relaxase enzyme. In the proposed experiments, the splicing-dependent conjugation system will be exploited in a combined molecular and genetic analysis of intron splicing. The specific aims include: - Use of simple genetic assays to isolate and characterize mutations affecting L1.1trB splicing. A major focus in this objective will be the intron sequences involved in interaction with the intron-encoded protein (IEP) and on the genetic and molecular basis of the maturase function conferred by the IEPs. - Analysis of the regulation of L1.1trB splicing functions, and examination of the extent to which splicing and conjugation functions are coordinately regulated. - Identification and analysis of small molecule splicing inhibitors using a splicing-dependent conjugation assay.